Growth Hormone Receptor Regulation
Overview Growth Hormone is a vital hormone secreted by the somatotropic cells of the anterior pituitary gland, or adenohypophysis. GH, also known as somatotropin, initiates the growth, reproduction, and restoration of specific cells in humans and animals. Growth hormone performs a variety of other tasks in the body; it stimulates production of IGF-1 by the liver, increases calcium retention, and promotes lipolysis and protein synthesis. Excess GH causes gigantism if the condition is experienced prior to the closing of bone epiphsyses and acromegaly if the condition occurs after the closing of bone epiphyses, which usually happens during puberty. Growth Hormone Receptor (GHR) is the binding site for GH on target cells; insufficient GHR is associated with growth and metabolic disorders while excess GHR is representative of some cancers. GHR is the gene that encodes for the receptor protein, and it is located on the 5th chromosome at 5p13-p12 and spans about 300kb. GH functions by binding to the high affinity GHR, which is phosphorylated along with associated tyrosine kinases initiating a intracellular signalling cascade that concludes with the biological functions of GH. Regulatory Mechanisms The ability of GH to perform its functions on target is directly related to the amount of functional GHRs on a target cell. Insufficient amounts of GHR lead to a variety of comorbidities such as decreased bone density, lipid disorders, and osteoporosis. Understanding the function of GHR regulation allows us gain targets for potential therapies, and test synthetic agents of regulation. Regulation with miRNAs It is known that GHR expression can be regulated at the primers of the 5'-UTR, untranslated region, of the mRNA. One study focused on the novel regulation of the 3'-UTR of mRNA with microRNAs. miRNAs are small non-coding RNA strands that function in gene silencing and post-transcriptional regulation of gene expression, and they typically effect the 3'-UTR making them the perfect fit for this experimental regulation. They function as part of ribonucleoprotein complexes referred to miRNA-induced silencing complexes, which prevent expression via mRNA degradation or inhibition of translation/translation machinery. Three cell types were used in the experiment, the primary model was the human embryonic cell line HEK293, and then two cancer cell lines MCF7 (mammary gland/breast) and LNCaP (prostate). The cancer cell lines were used because GHR mRNA is shown to increase 2-5 fold in a number of carcinomas including breast and prostate carcinomas when compared to their normal corresponding tissues. There are over two thousand mature miRNAs that exist in the body that could theoretically be purified, and so it was not definitive which miRNAs have the highest affinity for the GHR 3'-UTR. The binding specificity is a challenge with multiple cell lines as well, and so multiple strands of miRNAs were used to measure expression they are: miR-129-5p, miR-142-3p, miR-202, and miR-16. The cell lines were transfected with the miRNAs and after an incubation period the relative GHR mRNA levels were valued using PCR. The results of the study indicated statistically significant decreases in GHR mRNA and GHR protein after transfection with the miRNAs. -In the HEK293 cell line the level of GHR mRNA was reduced by 60% with miR-129-5p or miR-142-3p and about 30% with the other miRNAs. The level of GHR protein was reduced about 40% with 129-5p or 142-3p and by about 30% with miR-202 and miR-16. -For the LNCaP cell line the level of GHR mRNA was reduced by 43-60% with miR129-5p, miR-202, or miR-16, but miR-142-3p had no affect. The GHR protein levels decreased from 35%-74% for all of the miRNAs. -The transfection of the MCF7 cell line lead to decreases of GHR mRNA 30-70% and GHR protein decrease of about 35% with miR129-5p, miR142-3p, and miR-202. But with miR-16 there was a significant decrease in GHR protein but not mRNA suggesting that miR-16 acts in degradation during the translation process. The results from the miRNA transfection represent our ability to regulate genes at various levels of repression. Using synthetic miRNAs to regulate GHR levels and purified GH to ensure regular secretion we could fine tune the function of GH in the body of a patient who has expressed complications in the process. Natural Regulation Normal body fat mass accumulation occurs because of nutritional and environmental cues stimulate adipose tissue hyperplasia and hypertrophy. Adipocytes are one of the main targets of GH, which stimulate adipogenesis, lipogenesis, and lipolysis. It was thought that idiopathic obesity then could be caused in part due to GH insensitivity. GH treatment has had positive effects on body fat mass of individuals with endocrinopathy-related obesity, it has shown no results on those with idiopathic obesity. The main factor contributing to these results is because it has been measured that obese individuals have significantly lower GHR mRNA levels than fit individuals. This data suggests that GH resistance obesity is more a result of insufficient GHR levels rather than a GH deficiency or malfunction. Another characteristic associated with obesity is increased adipose levels of tumor-necrotic factor alpha (TNFa) due to increased macrophage infiltration of tissue and increased intracellular cortisol, the most vital human glucocorticoid, levels causing the increased presence of dehydrogenase enzymes. One study illustrated that increased TNFa levels are involved in the repression of GHR mRNA synthesis. The importance of this study is that it depicts a decrease in gene expression due to altered nutrition and conditions, and this decreased expression perpetuates the disease state that caused the deficiency. The information allows us to target a specific set of genes, which through up-regulation could possibly reverse the disease state. This mechanism of identifying specific protein deficiencies due to disease state cofactors is key for therapy of idiopathic diseases. The measurement of specific GHR mRNA and GHR protein is a technique that allows researchers to identify the underlying causes of disease that may not have been apparent before hand. The basis of measuring both specific mRNA and subsequent proteins allows identification of the exact point in either transcription or translation processes in which a deformation is occuring. Only by identifying specific binding points and locations of mutations can targets be chosen for molecular drugs. References NCBI Gene: GHR Erman, A. Wabitsch, M (2011): Human growth hormone receptor expression in obesity Elzein, S. Goodyer, CG (2014): Regulation of human growth hormone receptor expression with microRNAs